Trypanosoma cruzi, a Stercorarian trypanosome, causes Chagas' disease, a chronic illness affecting over 20 million people in Central and South America. This obligate intracellular parasite has a complex life cycle, with multiple developmental stages in the insect vector and the mammalian host. The immune system of the host mounts a strong response to the organisms, but sterile immunity is rarely if ever achieved, as the parasite lies protected in the cytoplasm of host cells. GP72 is a 72 kDa immunodominant surface glycoprotein, supposedly expressed only in the insect stages (epimastigotes and metacyclic trypomastigotes) of T. cruzi. Because the monoclonal antibody (WIC 29.26) that defined GP72 recognizes carbohydrate epitopes, the question of whether the gene is expressed in other stages, but differently posttranslationally modified, has not been resolved. GP72 is now one of the best defined of all the described T. cruzi surface antigens. There are some indications of possible functions of GP72. It is a major acceptor for C3, it is very immunogenic, and GP72- immunized mice are resistant to challenge by metacyclic trypomastigotes. However, the purity of GP72 previously used for immunization studies is questionable. GP72 has been purified and its gene has been cloned and sequenced. T. cruzi Y strain contains a single pair of GP72 genes and a Bull mutant has been constructed by sequential deletion of GP72 genes. These studies showed that the WIC 29.26 monoclonal antibody recognizes glycoconjugates other than GP72. The null mutant has a striking morphological phenotype, involving flagellar detachment. Morphological abnormalities persist into the infective trypomastigote stages, providing the best evidence so far that GP72 is normally expressed in these stages. The aim of this proposal is to further study the structure, expression, cellular location and biological function of GP72, its gene, and its novel attached glycans. Investigations will determine the structure and location of the attached glycans. The role of GP72 and the WIC 29.26 glycans in colonization of the insect vector, which is impaired in the null mutant, will be further studied. The approaches used will utilize our recently developed ability to perform sophisticated genetic manipulations of trypanosomatid protozoa. For example, epitope tagging will provide a marker to clarify both the cellular location and the stage-specific expression of the GP72 polypeptide, which could not be proven by existing methods. Using the cloned gene and antibodies raised against the corresponding protein, or a tagged variant, we will study the kinetics of biosynthesis and posttranslational modification of GP72. From the knowledge gained from structural studies of GP72 during the life cycle, the immunoprophylactic potential of recombinant proteins and synthetic glycopeptides will be evaluated.